Disease control with tick phospholipase A2

ABSTRACT

The present invention relates to reagents and methods for the modulation of viability of bacteria. A process is provided wherein a protein sequence from  A. americanum  saliva effective in reducing the viability of gram positive, gram negative, or acid-fast bacteria and spirochetes including  B. burgdurferi  is administered. The inventive protein from  A. americanum  saliva has utility as a therapeutic for the treatment of an organism infected with bacteria, particularly the spirochete  B. burgdorferi.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional Application No.61/143,304 filed Jan. 8, 2009, and is the U.S. National Stageapplication of PCT/US2010/020513, the entire contents of each of whichare incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates generally to the field of pharmaceuticaland diagnostic compositions useful in the diagnosis, treatment andprophylaxis of Lyme borreliosis. More specifically the present inventionrelates to methods of preparation of, sequence relating to, and use ofphospholipase A₂ or phospholipase A₂-like protein derived from the tickAmblyomma americanum.

BACKGROUND OF THE INVENTION

Borrelia burgdorferi (sensu lato) encompasses several Borrelia speciesbelieved to be the causative agent of Lyme borreliosis (Lyme disease)including: B. burgdorferi sensu strict; B. garinii; and B. afzelii. Lymedisease is transmitted by the bite of various species of Ixodes tickscarrying the spirochete. The main reservoir of the infection in theUnited States is the white footed mouse, Peromyscus leucopus, and theinfection can be transmitted to many mammalian species including dogs,cats, and man. (J. G. Donahue, et al, Am. J. Trop. Med. Hyg., 36:92-96(1987); R. T. Green, et al, J. Clin. Micro., 26:648-653 (1988)).

Amblyomma americanum (Linnaeus), the lone star tick, is the primaryvector of Ehrlichia chaffeensis, the bacterial agent of human monocyticehrlichiosis (Anderson et al., 1993). In addition to Ehrlichiachaffeensis this aggressive human-biting tick species is also thought tovector other potential pathogens including Ehrlichia ewingii, Rickettsiarickettsii, R. amblyommii, Borrelia lonestari, Francisella tularensisand Coxiella burnetii (Calhoun, 1954; Burgdorfer, 1975; Barbour et al.,1996; Murphy et al., 1998; Burkot et al., 2001; Childs & Paddock, 2003).While Amblyomma americanum is found throughout geographical regions witha high occurrence of Lyme disease (Bishopp & Trembley, 1945; Anderson &Magnarelli, 1980; Hair & Bowman, 1986; Ginsberg et al., 1991; Luckhartet al., 1991; Keirans & Lacombe, 1998), there is no evidence that A.americanum transmits B. burgdorferi (Piesman & Happ, 1997). Reasonsexplaining why A. americanum fails to act as a suitable vector for B.burgdorferi have remained elusive.

Generally, arthropod vector refractoriness to a pathogen can beclassified into three categories: (1) a lack of acquisition; (2) aninability to maintain growth of the pathogen; or (3) an inability totransmit the pathogen. In controlled experimental studies, as many as25% of A. americanum larvae acquired spirochetes during infectiousfeedings. However, nearly all of these larvae were spirochete-negativebefore moulting to the nymphal stage (Piesman & Sinsky, 1988; Mather &Mather, 1990; Ryder et al., 1992; Piesman & Happ, 1997). Indeed, therefractory nature of A. americanum to maintaining and transmittingspirochetes has been previously recognized. Only one report oftrans-stadial maintenance of infection in A. americanum nymphs has beenpublished (Ryder et al., 1992), and one report of spirochete isolatesfrom three pools of A. americanum adults (Teltow et al., 1991). However,these studies predate the discovery of the spirochete B. lonestari, orused methods that would not be able to discriminate between the twospirochete species, B. burgdorferi and B. lonestari (Barbour et al.,1996). Importantly, there has been no successful reported transmissionof B. burgdorferi between infected and naïve hosts by A. americanum(Mukolwe et al., 1992; Ryder et al., 1992; Oliver et al., 1993; Sanders& Oliver, 1995; Piesman & Happ, 1997).

During A. americanum, I. scapularis and other Ixodid tick feeding,saliva is continuously secreted into the host (McMullen & Sauer, 1978;Coons et al., 1986; Sonenshine, 1991). A possible mechanism regulatingtransfer of B. burgdorferi has been previously identified as a factor inthe saliva of A. americanum. (Ledin, K. E., et al., Med. Vet. Entomol.,2005; 19(1):90-95. However, specific molecules responsible forpreventing transmission of B. burgdorferi during tick feeding areunknown making design and implementation of therapeutic methods andreagents difficult. Thus, there exists a need for identification ofagents capable of controlling infection, growth, and viability of B.burgdorferi.

SUMMARY OF THE INVENTION

The present invention provides process for modulating a bacterium byadministering a salivary protein of the tick A. americanum withphospholipase A₂-like activity. The protein preferably has a migratorymolecular weight between 53 and 69 kDa. Several bacterial organisms aremodulated by the invention. Preferably the bacterial organism is amember of the Spriochaetaceae family or the Treponemataceae family. Morepreferably, the bacterial organism is Borrelia burgdorferi, Borreliacrocidurae, Borrelia lusitaniae, Borrelia recurrentis, Borrelia hermsii,Borrelia parkeri, Borrelia lonestari, Borrelia afzelii, Borreliagarinii, Borrelia recurrentis, Borrelia buccalis, Borrelia refringens,S. aureus, E. coli, L. monocytogenes, S. choleraesuis, S. typhi, S.enteritidis, S. pullorum, Bacillus anthracis, or M. tuberculosis.

A therapeutic is provided that includes a suitable pharmaceuticalcarrier and a phospholipase A₂-like protein derived from A. americanum.The therapeutic is operable for treating Lyme disease in a vertebratehost. As such an inventive process of treating Lyme disease in avertebrate host or modulating a Borrelia burgdorferi infection isprovided including administering the therapeutic including thephospholipase A₂-like protein derived from A. americanum. The inventivetherapeutic is administered at concentrations from about 0.05 to about10,000 micrograms/milliliter. These concentrations are optionallymaximal plasma concentrations. The inventive process optionally includesadministering the therapeutic between one and three times per day.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents fluorescent microscopy indicating live (SYTO9, white)and dead (propidium iodide, gray) staining as a measure of B.burgdorferi survival 48 h after exposure to specific in vitro treatments(100× magnification). (A) PBS, (B) I. scapularis saliva, (C)Pilocarpine, (D) Phospholipase A₂ derived from A. americanum saliva;

FIG. 2 represents (A) Mean live B. burgdorferi counts per 10 highpowered (100×) fields and estimated 95% confidence intervals afterexposure to phospholipase A₂ derived from A. americanum saliva,pilocarpine, I. scapularis saliva, or PBS at 24 and 48 h post-exposure;

FIG. 3 represents elimination of borreliacidal activity in A. americanumsaliva after incubation with trypsin;

FIG. 4 represents comparative protein profiles of A. americanum and I.scapularis saliva as demonstrated by PAGE;

FIG. 5 represents (A) Borreliacidal activity of pools (1-11) of A.americanum saliva after fractionation by gel filtration HPLC and (B)Borreliacidal activity of A. americanum saliva fractions;

FIG. 6 represents (A) two dimensional gel electrophoresis of activeborreliacidal fractions of A. americanum saliva and (B) two dimensionalgel electrophoresis of inactive fractions of A. americanum saliva;

FIG. 7 represents inhibition of the borreliacidal activity of A.americanum saliva after incubation with oleyloxyethyl phosphorylcholine(OPC).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention describes a novel nucleotide encoding a proteinthat resembles phospholipase A₂ from the tick A. americanum, and theprotein derived therefrom, that has effectiveness in modulating anactivity of a bacterial organism, illustratively, altering the viabilityof spirochetes and other bacterial organisms. Illustratively, theinvention regulates viability of the Lyme disease spirochete B.burgdorferi or bacteria illustratively including S. aureus, E. coli, andL. monocytogenes. Thus, the invention has utility in regulatingviability of spirochetes and bacteria.

The following description of the preferred embodiment(s) is merelyexemplary in nature and is in no way intended to limit the scope of theinvention, its application, or uses. The invention is described withrelation to the non-limiting definitions included herein. Thesedefinitions are not designed to function as a limitation on the scope orpractice of the invention but are presented for illustrative anddescriptive purposes only.

The effectiveness of a phospholipase A₂-like protein (PLA2) inmodulating B. burgdorferi viability was unexpected given that PLA2preferably has a migratory molecular weight between 53 and 69 kDa. Morepreferably PLA2 has a migratory molecular weight between 58 and 62 kDa.It is appreciated that an inventive PLA2 protein is optionally monomericor polymeric. Optionally the PLA2 protein is monomeric, dimeric,trimeric, pentameric, or hexameric. Previously described prior artphospholipase A₂ molecules have much lower molecular weights commonlybetween 14-15 kDa. While the mechanism may not be entirely tied to theprotein length or molecular weight and lower molecular weight fragmentsof PLA2 are similarly operable, the mechanism of action of the A.americanum PLA2 of the present invention has unique functionalproperties that distinguish it from its mammalian counterparts allowingfor low toxicity and cross reactivity when administered to a subjectsuch as to treat a disease or infection.

The present invention provides methods and compositions for treatingconditions or disorders having a relationship to infection of anorganism by a spirochete or other bacteria. Illustrative examples ofinfectious agents addressed by administration or exposure to aninventive compound or material include those of the Spirochaetaceaefamily including the Borrellia genus and the Treponema genus.Illustratively organisms that may be targeted by the inventive compoundor method include B. burgdorferi, Borrelia crocidurae, Borrelialusitaniae, Borrelia recurrentis, Borrelia hermsii, Borrelia parkeri,Borrelia lonestari, Borrelia afzelii, Borrelia garinii, Borreliarecurrentis, Borrelia buccalis, and Borrelia refringens. B. burdorferiis most preferred.

Members of the genus Leptospira are similarly amenable to modulation bythe inventive compound. Illustratively, serovars Icterohaemorrhagiae,Canicola, Pomona, Grippotyphosa, and Bratislava are amenable tomodulation.

It is recognized that many other members of the Spriochaetaceae family,the Treponemataceae family, and other bacterial organisms are known andare recognized as similarly targetable by the present invention. Membersof human pathogen groups Leptospira (non-limiting ex. L. interrogans, L.canicol, L. biflexia), Borrelia, and Treponema are particularlypreferred.

Non spirochete organisms are similarly targetable by the presentinvention. Organisms illustratively include S. aureus, E. coli, L.monocytogenes, S. choleraesuis, S. typhi, S. enteritidis, S. pullorum,Bacillus anthracis, M. tuberculosis, and other gram positive bacteria.

Diseases caused by agents that may be illustratively modulated by thepresent invention include but are not limited to Lyme disease, relapsingfever, leptospirosis, rat bite fever, Vincent's angina, syphilis, yaws,pinta, periodontal disease, oral soft tissue infections, fusospirochetaldisease, conjunctivitis, septicemia, granulomatosis infantisepticum,listeriosis, tuberculosis, and anthrax.

As used herein, the terms “subject” or “organism” are treatedsynonymously and are defined as any organism capable of hostinginfection of a spirochete or bacteria. A subject illustratively includesa mammal, humans, non-human primates, horses, goats, cows, sheep, pigs,dogs, cats, rodents, arthropods, ticks, and cells.

A therapeutically effective amount is defined as an amount of aninventive compound that when administered to a subject, ameliorates acondition or symptom of infection.

The terms “biologically active peptide” and “peptide therapeutic agent,”“peptide,” and “protein” are synonymous as used herein and are intendedto mean a natural or synthetic compound containing two or more aminoacids, particularly protein that participates in modulating an aspect ofa target organism, illustratively, a bacterial organism. It isappreciated that a protein optionally has fewer or more amino acids thanthe wild-type sequence. Amino acids present in a protein illustrativelyinclude the common amino acids alanine, cysteine, aspartic acid,glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine,leucine, methionine, asparagine, proline, glutamine, arginine, senile,threonine, valine, tryptophan, and tyrosine as well as less commonnaturally occurring amino acids, modified amino acids or syntheticcompounds, such as alpha-asparagine, 2-aminobutanoic acid or2-aminobutyric acid, 4-aminobutyric acid, 2-aminocapric acid(2-aminodecanoic acid), 6-aminocaproic acid, alpha-glutamine,2-aminoheptanoic acid, 6-aminohexanoic acid, alpha-aminoisobutyric acid(2-aminoalanine), 3-aminoisobutyric acid, beta-alanine,allo-hydroxylysine, allo-isoleucine, 4-amino-7-methylheptanoic acid,4-amino-5-phenylpentanoic acid, 2-aminopimelic acid,gamma-amino-beta-hydroxybenzenepentanoic acid, 2-aminosuberic acid,2-carboxyazetidine, beta-alanine, beta-aspartic acid, biphenylalanine,3,6-diaminohexanoic acid, butanoic acid, cyclobutyl alanine,cyclohexylalanine, cyclohexylglycine, N5-aminocarbonylornithine,cyclopentyl alanine, cyclopropyl alanine, 3-sulfoalanine,2,4-diaminobutanoic acid, diaminopropionic acid, 2,4-diaminobutyricacid, diphenyl alanine, N,N-dimethylglycine, diaminopimelic acid,2,3-diaminopropanoic acid, S-ethylthiocysteine, N-ethylasparagine,N-ethylglycine, 4-aza-phenylalanine, 4-fluoro-phenylalanine,gamma-glutamic acid, gamma-carboxyglutamic acid, hydroxyacetic acid,pyroglutamic acid, homoarginine, homocysteic acid, homocysteine,homohistidine, 2-hydroxyisovaleric acid, homophenylalanine, homoleucine,homoproline, homoserine, homoserine, 2-hydroxypentanoic acid,5-hydroxylysine, 4-hydroxyproline, 2-carboxyoctahydroindole,3-carboxyisoquinoline, isovaline, 2-hydroxypropanoic acid (lactic acid),mercaptoacetic acid, mercaptobutanoic acid, sarcosine,4-methyl-3-hydroxyproline, mercaptopropanoic acid, norleucine, nipecoticacid, nortyrosine, norvaline, omega-amino acid, ornithine, penicillamine(3-mercaptovaline), 2-phenylglycine, 2-carboxypiperidine, sarcosine(N-methylglycine), 2-amino-3-(4-sulfophenyl)propionic acid,1-amino-1-carboxycyclopentane, 3-thienylalanine,epsilon-N-trimethyllysine, 3-thiazolylalanine, thiazolidine 4-carboxylicacid, alpha-amino-2,4-dioxopyrimidinepropanoic acid, and2-naphthylalanine. Accordingly, the term “biologically active peptide”as used herein includes peptides having between 2 and about 1000 aminoacids or having a molecular weight in the range of about 150-100,000Daltons.

A biologically active peptide is obtained by any of various methodsknown in the art illustratively including isolation from a cell ororganism, chemical synthesis, expression of a nucleic acid and partialhydrolysis of proteins. Chemical methods of peptide synthesis are knownin the art and include solid phase peptide synthesis and solution phasepeptide synthesis for instance. A biologically active peptide includedin an inventive composition may be a naturally occurring ornon-naturally occurring peptide. The term “naturally occurring” refersto a peptide endogenous to a cell, tissue or organism and includesallelic variations. A non-naturally occurring peptide is synthetic orproduced apart from its naturally associated organism or modified and isnot found in an unmodified cell, tissue or organism.

The term “biological activity” as used herein is intended to mean anactivity usually associated with a peptide or nucleic acid. Biologicalactivity includes activity described at a molecular level such asreceptor binding/blocking, receptor activation/inhibition, ion channelmodulation, second messenger modulation, and membrane disruption.Biological activity further includes activity described at a cellular orsubcellular level such as stimulation/inhibition of synaptic release. Inaddition, biological activity further includes activity described at anorganismal level such as behavioral changes, changes in perception ofpain, and decreased nausea and/or vomiting. Biological activity of apeptide is measurable and may be assessed by techniques known in theart.

As used herein, the term “sample” is defined as material obtained from abiological organism, tissue, cell, cell culture medium, or any mediumsuitable for mimicking biological conditions, or from the environment.Non-limiting examples include, saliva, gingival secretions,cerebrospinal fluid, gastrointestinal fluid, mucous, urogenitalsecretions, synovial fluid, cerebrospinal fluid, blood, serum, plasma,urine, cystic fluid, lymph fluid, ascites, pleural effusion,interstitial fluid, intracellular fluid, ocular fluids, seminal fluid,mammary secretions, vitreal fluid, nasal secretions, water, air, gas,powder, soil, biological waste, feces, cell culture media, cytoplasm,cell releasate, cell lysate, buffers, or any other fluid or solid media.

The term “nucleotide” is intended to mean a base-sugar-phosphatecombination either natural or synthetic, linear, circular and sequentialarrays of nucleotides and nucleosides, e.g. cDNA, genomic DNA, mRNA, andRNA, oligonucleotides, oligonucleotides, and derivatives thereof.Included in this definition are modified nucleotides which includeadditions to the sugar-phosphate groups as well as to the bases.

The term “nucleic acid” or “polynucleotide” refers to multiplenucleotides attached in the form of a single or double strandedpolynucleotide that can be natural, or derived synthetically,enzymatically, and by cloning methods. The term “oligonucleotide” refersto a polynucleotide of less than 200 nucleotides. The terms “nucleicacid” and “oligonucleotide” may be used interchangeably in thisapplication.

An inventive nucleic acid sequence is provided. The nucleic acidsequence relates to the gene encoding PLA2 derived from the tick A.americanum. The nucleic acid sequence preferably encodes salivary PLA2derived from the tick A. americanum. The inventive nucleic acid sequenceis preferably isolated from the cellular materials with which it isnaturally associated. It is appreciated that a nucleic acid sequencethat hybridizes with a nucleic acid sequence encoding PLA2 protein iswithin the scope of the invention. A hybridizing nucleic acid sequenceoptionally has a sequence 100 percent complementary to a nucleic acidsequence encoding PLA2 or a portion thereof. Optionally, a hybridizingnucleic acid sequence has 50, 60, 70, 80, 90, 95, 99, or greatercomplementarity to a nucleic acid sequence encoding PLA2 or a fragmentthereof.

As used herein, the term “hybridizes” preferably describes hybridizationunder stringent conditions for hybridization and washing under whichnucleotide sequences having at least 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or greater complementarity toeach other typically remain hybridized to each other. Such hybridizationconditions are described in, for example but not limited to, CurrentProtocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.16.3.6.; Basic Methods in Molecular Biology, Elsevier Science PublishingCo., Inc., N.Y. (1986), pp. 75 78, and 84 87; and Molecular Cloning,Cold Spring Harbor Laboratory, N.Y. (1982), pp. 387 389, and are wellknown to those skilled in the art. A preferred, non-limiting example ofstringent hybridization conditions is hybridization in 6× sodiumchloride/sodium citrate (SSC), 0.5% SDS at about 68° C. followed by oneor more washes in 2×SSC, 0.5% SDS at room temperature. Anotherpreferred, non-limiting example of stringent hybridization conditions ishybridization in 6×SSC at about 45° C. followed by one or more washes in0.2×SSC, 0.1% SDS at 50 to 65° C.

Numerous methods are known in the art for the synthesis and productionof nucleic acid sequences illustratively including cloning andexpression in cells such as E. coli, insect cells such as Sf9 cells,yeast, and mammalian cell types such as Hela cells, Chinese hamsterovary cells, or other cell systems known in the art as amenable totransfection and nucleic acid and/or protein expression. Methods ofnucleic acid isolation are similarly recognized in the art.Illustratively, plasmid DNA amplified in E. coli is cleaved by suitablerestriction enzymes such as NdeI and XhoI to linearize PLA2 DNA. ThePLA2 DNA is subsequently isolated following gel electrophoresis using aS.N.A.P.™ UV-Free Gel Purification Kit (Invitrogen, Carlsbad, Calif.) asper the manufacturer's instructions.

Numerous agents are amenable to facilitate cell transfectionillustratively including synthetic or natural transfection agents suchas LIPOFECTIN, baculovirus, naked plasmid or other DNA, or other systemsknown in the art.

The present invention includes variants of PLA2 such as allelicvariants, mutational variants, insertional variants, deletion variants,or nucleotide variants illustratively including derivative nucleotidesand amino acid variants. As used herein, the term “variant” defineseither a naturally occurring genetic mutant of PLA2, or a recombinantlyprepared variation of PLA2, each of which contain one or more mutationsin its genome compared to the wild type PLA2. A variant optionallyincludes a derivative.

As used herein, the term “derivative” in the context of anon-proteinaceous derivative defines a second organic or inorganicmolecule that is formed based upon the structure of a first organic orinorganic molecule. A derivative of an organic molecule includes, but isnot limited to, a molecule modified, e.g., by the addition or deletionof a hydroxyl, methyl, ethyl, carboxyl or amine group. An organicmolecule may also be esterified, alkylated and/or phosphorylated. Aderivative also defined as a degenerate base mimicking a C/T mix such asthat from Glen Research Corporation, Sterling, Va., illustrativelyLNA-dA or LNA-dT, or other nucleotide modification known in the art orotherwise. A nucleotide is optionally locked.

A nucleotide sequence variant is preferably greater than 50 percentidentical to a nucleic acid sequence encoding PLA2 or a fragmentthereof. More preferably, a nucleotide sequence variant is greater than75 percent identical to a nucleic acid sequence encoding PLA2 or afragment thereof. A nucleotide sequence variant is preferably 80, 85,90, 95, 99 percent identical or greater to a nucleic acid sequenceencoding PLA2 or a fragment thereof.

Similarly, an amino acid sequence variant is preferably greater than 50percent identical to an amino acid sequence of PLA2 or a fragmentthereof. More preferably, an amino acid sequence variant is greater than75 percent identical to an amino acid sequence of PLA2 or a fragmentthereof. An amino acid variant is preferably 80, 85, 90, 95, 99 percentidentical or greater to an amino acid sequence of PLA2 or a fragmentthereof.

It is recognized that several forms of phospholipase A₂ from numerousorganisms exist as allelic variants. Illustratively, human lipoproteinassociated phospholipase A₂ is found with known variants of −1357G>A,-403T>C, and variants producing amino acid substitutions Arg92His,Ile198Thr, Ala379Val. (Hoffmann, M M, et al., J Thromb Haemost., 2009;7(1):41-8) Similarly, allelic variants of PLA2 are recognized and withinthe scope of the present invention.

The nucleotide sequences of the invention are optionally isolated byconventional uses of polymerase chain reaction or cloning techniquessuch as those described in conventional texts. For example, the nucleicacid sequences of this invention are optionally prepared or isolatedfrom DNA using DNA primers and probes and PCR techniques. Alternatively,the inventive PLA2 nucleic acid sequence is obtained from gene banksderived from A. americanum whole genomic DNA. These sequences, fragmentsthereof, modifications thereto and the full-length sequences areoptionally constructed recombinantly using conventional geneticengineering or chemical synthesis techniques or PCR, and the like.

Modulation of bacterial target activity is illustratively accomplishedwith a fragment of PLA2. As the molecular weight of the protein encodedby the a preferred embodiment of isolated PLA2 nucleotide sequence is58-62 kDa and other forms of phospholipase A2 commonly range from 14-15kDa, functional fragments of PLA2 are recognized as having activitytoward modulating an aspect of a target organism such as viability.

As used herein the term “modulating” refers to altering a function,cycle, characteristic, or target of an infectious agent. Preferably,modulating is altering the viability of an organism. Modulating isoptionally alteration of transcription, translation, molecularlocalization, modification, or activity.

The present invention also encompasses an isolated phospholipase A₂protein derived from A. americanum. In a preferred embodiment aninventive PLA2 protein has the sequence represented by an amino acidsequence of PLA2 or a fragment thereof. PLA2 protein is preferablyrecombinant. However, it is also envisioned that naturally occurringPLA2 protein is optionally isolated from at least a portion of thecellular and other sample material for which the wild type sequence isnormally found. Methods for purification of protein from organismderived samples are known and are within the level of skill in the art.

Modifications and changes can be made in the structure of thephospholipase A₂ protein derived from A. americanum that is the subjectof the application to create a variant thereof and still obtain amolecule having similar characteristics as the phospholipase (e.g., aconservative amino acid substitution). For example, certain amino acidscan be substituted for other amino acids in a sequence withoutappreciable loss of activity. Because it is the interactive capacity andnature of a polypeptide that defines that polypeptide's biologicalfunctional activity, certain amino acid sequence substitutions can bemade in a polypeptide sequence and nevertheless obtain a polypeptidewith like properties.

In making such changes, the hydropathic index of amino acids can beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a polypeptide is generallyunderstood in the art. It is known that certain amino acids can besubstituted for other amino acids having a similar hydropathic index orscore and still result in a polypeptide with similar biologicalactivity. Each amino acid has been assigned a hydropathic index on thebasis of its hydrophobicity and charge characteristics. Those indicesare: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenyl alanine(+2.8); cysteine/cysteine (+2.5); methionine (+1.9); alanine (+1.8);glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9);tyrosine (−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5);glutamine (−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9);and arginine (−4.5).

It is believed that the relative hydropathic character of the amino aciddetermines the secondary structure of the resultant polypeptide, whichin turn defines the interaction of the polypeptide with other molecules,such as enzymes, substrates, receptors, antibodies, antigens, and thelike. It is known in the art that an amino acid can be substituted byanother amino acid having a similar hydropathic index and still obtain afunctionally equivalent polypeptide. In such changes, the substitutionof amino acids whose hydropathic indices are within ±2 is preferred,those within ±1 are particularly preferred, and those within ±0.5 areeven more particularly preferred.

Substitution of like amino acids can also be made on the basis ofhydrophilicity, particularly, where the biological functional equivalentpolypeptide or peptide thereby created is intended for use inimmunological embodiments. The following hydrophilicity values have beenassigned to amino acid residues: arginine (+3.0); lysine (+3.0);aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine(+0.2); glutamine (+0.2); glycine (0); proline (−0.5±1); threonine(−0.4); alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine(−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine(−2.3); phenylalanine (−2.5); tryptophan (−3.4). It is understood thatan amino acid can be substituted for another having a similarhydrophilicity value and still obtain a biologically equivalent, and inparticular, an immunologically equivalent polypeptide. In such changes,the substitution of amino acids whose hydrophilicity values are within±2 is preferred, those within ±1 are particularly preferred, and thosewithin ±0.5 are even more particularly preferred.

As outlined above, amino acid substitutions are generally based on therelative similarity of the amino acid side-chain substituents, forexample, their hydrophobicity, hydrophilicity, charge, size, and thelike. Exemplary substitutions that take various of the foregoingcharacteristics into consideration are well known to those of skill inthe art and include (original residue: exemplary substitution): (Ala:Gly, Ser), (Arg: Lys), (Asn: Gln, His), (Asp: Glu, Cys, Ser), (Gln:Asn), (Glu: Asp), (Gly: Ala), (His: Asn, Gln), (Ile: Leu, Val), (Leu:Ile, Val), (Lys: Arg), (Met: Leu, Tyr), (Ser: Thr), (Thr: Ser), (Tip:Tyr), (Tyr: Trp, Phe), and (Val: Ile, Leu). Embodiments of thisdisclosure thus contemplate functional or biological equivalents of apolypeptide as set forth above. In particular, embodiments of thepolypeptides can include variants having about 50%, 60%, 70%, 80%, 90%,and 95% sequence identity to the polypeptide of interest.

An inventive PLA2 protein is illustratively recombinant. An inventiveprotein is optionally coexpressed with associated tags, modifications,other proteins such as in a fusion peptide, or other modifications orcombinations recognized in the art. Illustrative tags include 6× His,FLAG, biotin, ubiquitin, SUMO, or other tag known in the art. A tag isillustratively cleavable such as by linking to PLA2 or an associatedprotein via an enzyme cleavage sequence that is cleavable by an enzymeknown in the art illustratively including Factor Xa, thrombin, SUMOstarprotein as obtainable from Lifesensors, Inc., Malvern, Pa., or trypsin.It is further appreciated that chemical cleavage is similarly operablewith an appropriate cleavable linker.

Protein expression is illustratively accomplished from transcription ofPLA2 nucleic acid sequence, translation of RNA transcribed from PLA2nucleic acid sequence, modifications thereof, or fragments thereof.Protein expression is preferably performed in a cell based system suchas in E. coli, Hela cells, insect cells, or Chinese hamster ovary cells.Eukaryotic, are preferred. Insect cells are particularly preferred. Itis appreciated that cell-free expression systems are similarly operable.

It is recognized that numerous variants, analogues, or homologues arewithin the scope of the present invention including amino acidsubstitutions, alterations, modifications, deletions, insertions, orother amino acid changes that increase, decrease, or do not alter thefunction of the PLA2 protein sequence. Several post-translationalmodifications are similarly envisioned as within the scope of thepresent invention illustratively including incorporation of anon-naturally occurring amino acid, phosphorylation, glycosylation,addition of pendent groups such as biotin, fluorophores, lumiphores,radioactive groups, antigens, or other molecules.

The present invention also provides a vector with an inventive PLA2sequence therein. Illustrative vectors include a plasmid, cosmid,cationic lipids, non-liposomal cationic vectors, cationic cyclodextrin,viruses with RNA or DNA genetic material, polyethylenimines,histidylated polylysine, or other vector system known in the art. Avector is preferably a plasmid. A suitable vector optionally possessescell type specific expression or other regulatory sequences or sequencesoperable to stimulate or inhibit gene or protein expression. A vectorillustratively contains a selection marker such as an antibioticresistance gene.

Also provided is a host cell transformed with an appropriate vector orwith the inventive PLA2 sequence. A preferred host cell includes E. colior Sf9 cells. Optionally cell transfection is achieved byelectroporation.

A method is also provided for recombinantly expressing a inventive PLA2nucleic acid or protein sequence or fragments thereof wherein a cell istransformed with an inventive nucleic acid sequence and cultured undersuitable conditions that permit expression of PLA2 nucleic acid sequenceor protein either within the cell or secreted from the cell. Cellculture conditions are particular to cell type and expression vector.Culture conditions for particular vectors and cell types are within thelevel of skill in the art to design and implement without undueexperimentation.

Recombinant or non-recombinant proteinase peptides or recombinant ornon-recombinant proteinase inhibitor peptides or other non-peptideproteinase inhibitors can also be used in the present invention.Proteinase inhibitors are optionally modified to resist degradation, forexample degradation by digestive enzymes and conditions. Techniques forthe expression and purification of recombinant proteins are known in theart (see Sambrook Eds., Molecular Cloning: A Laboratory Manual 3^(rd)ed. (Cold Spring Harbor, N.Y. 2001).

Some embodiments of the present invention are compositions containingPLA2 nucleic acid that can be expressed as encoded polypeptides orproteins. The engineering of DNA segment(s) for expression in aprokaryotic or eukaryotic system may be performed by techniquesgenerally known to those of skill in recombinant expression. It isbelieved that virtually any expression system is operable for theexpression of the claimed nucleic and amino sequences.

Generally speaking, it may be more convenient to employ as therecombinant polynucleotide a cDNA version of the polynucleotide. It isbelieved that the use of a cDNA version will provide advantages in thatthe size of the gene will generally be much smaller and more readilyemployed to transfect the targeted cell than will a genomic gene, whichwill typically be up to an order of magnitude larger than the cDNA gene.However, the inventor does not exclude the possibility of employing agenomic version of a particular gene where desired.

As used herein, the terms “engineered” and “recombinant” cells aresynonymous with “host” cells and are intended to refer to a cell intowhich an exogenous DNA segment or gene, such as a cDNA or gene has beenintroduced. Therefore, engineered cells are distinguishable fromnaturally occurring cells that do not contain a recombinantly introducedexogenous DNA segment or gene. A host cell is optionally a naturallyoccurring cell that is transformed with an exogenous DNA segment or geneor a cell that is not modified. A host cell preferably does not possessa naturally occurring gene encoding PLA2. Engineered cells are, thus,cells having a gene or genes introduced through the hand of man.Recombinant cells include those having an introduced cDNA or genomicDNA, and also include genes positioned adjacent to a promoter notnaturally associated with the particular introduced gene.

To express a recombinant encoded polypeptide in accordance with thepresent invention one illustratively prepares an expression vector thatincludes a polynucleotide under the control of one or more promoters. Tobring a coding sequence “under the control of” a promoter, one positionsthe 5′ end of the translational initiation site of the reading framegenerally between about 1 and 50 nucleotides “downstream” of (i.e., 3′of) the chosen promoter. The “upstream” promoter stimulatestranscription of the inserted DNA and promotes expression of the encodedrecombinant protein. This is the meaning of “recombinant expression” inthe context used here.

Many standard techniques are available to construct expression vectorscontaining the appropriate nucleic acids andtranscriptional/translational control sequences in order to achieveprotein or peptide expression in a variety of host-expression systems.Cell types available for expression include, but are not limited to,bacteria, such as E. coli and B. subtilis transformed with recombinantphage DNA, plasmid DNA or cosmid DNA expression vectors.

Certain examples of prokaryotic hosts are E. coli strain RR1, E. coliLE392, E. coli B, E. coli.chi. 1776 (ATCC No. 31537) as well as E. coliW3110 (F-, lambda-, prototrophic, ATCC No. 273325); bacilli such asBacillus subtilis; and other enterobacteriaceae such as Salmonellatyphimurium, Serratia marcescens, and various Pseudomonas species.

In general, plasmid vectors containing replicon and control sequencesthat are derived from species compatible with the host cell are used inconnection with these hosts. The vector ordinarily carries a replicationsite, as well as marking sequences that are capable of providingphenotypic selection in transformed cells. For example, E. coli is oftentransformed using pBR322, a plasmid derived from an E. coli species.Plasmid pBR322 contains genes for ampicillin and tetracycline resistanceand thus provides easy means for identifying transformed cells. ThepBR322 plasmid, or other microbial plasmid or phage preferably alsocontain, or be modified to contain, promoters that can be used by themicrobial organism for expression of its own proteins.

In addition, phage vectors containing replicon or control sequences thatare compatible with the host microorganism can be used as transformingvectors in connection with these hosts. For example, the phage lambda isoptionally utilized in making a recombinant phage vector that can beused to transform host cells, such as E. coli LE392.

Further useful vectors include pIN vectors and pGEX vectors, for use ingenerating glutathione S-transferase (GST) soluble fusion proteins forlater purification and separation or cleavage. Other suitable fusionproteins are those with β-galactosidase, ubiquitin, or the like.

Promoters that are most commonly used in recombinant DNA constructioninclude the β-lactamase (penicillinase), lactose and tryptophan (trp)promoter systems. While these are the most commonly used, othermicrobial promoters have been discovered and utilized, and detailsconcerning their nucleotide sequences have been published, enablingthose of skill in the art to ligate them functionally with plasmidvectors.

For expression in Saccharomyces, the plasmid YRp7, for example, isillustratively used. This plasmid contains the trp1 gene, which providesa selection marker for a mutant strain of yeast lacking the ability togrow in tryptophan, for example ATCC No. 44076 or PEP4-1. The presenceof the trp1 lesion as a characteristic of the yeast host cell genomethen provides an effective environment for detecting transformation bygrowth in the absence of tryptophan.

Suitable promoter sequences in yeast vectors include the promoters for3-phosphoglycerate kinase or other glycolytic enzymes, such as enolase,glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, and glucokinase. In constructing suitableexpression plasmids, the termination sequences associated with thesegenes are also ligated into the expression vector 3′ of the sequencedesired to be expressed to provide polyadenylation of the mRNA andtermination.

Other suitable promoters, which have the additional advantage oftranscription controlled by growth conditions, include the promoterregion for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase,degradative enzymes associated with nitrogen metabolism, and theaforementioned glyceraldehyde-3-phosphate dehydrogenase, and enzymesresponsible for maltose and galactose utilization.

In addition to microorganisms, cultures of cells derived frommulticellular organisms are also optionally used as hosts. In principle,any such cell is operable, whether from vertebrate or invertebrateculture. In addition to mammalian cells, these include insect cellsystems such as those infected with recombinant virus expression vectors(e.g., baculovirus); and plant cell systems such as those infected withrecombinant virus expression vectors (e.g., cauliflower mosaic virus,CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmidexpression vectors (e.g., Ti plasmid) containing one or more codingsequences.

In a useful insect system, Autographica californica nuclear polyhedrosisvirus (AcNPV) is used as a vector to express foreign genes. The virusgrows in Spodoptera frugiperda cells. The isolated nucleic acid codingsequences are cloned into non-essential regions (for example thepolyhedron gene) of the virus and placed under control of an AcNPVpromoter (for example, the polyhedron promoter). Successful insertion ofthe coding sequences results in the inactivation of the polyhedron geneand production of non-occluded recombinant virus (i.e., virus lackingthe proteinaceous coat coded for by the polyhedron gene). Theserecombinant viruses are then used to infect Spodoptera frugiperda cellsin which the inserted gene is expressed (e.g., U.S. Pat. No. 4,215,051incorporated herein by reference).

Examples of useful mammalian host cell lines are VERO and HeLa cells,Chinese hamster ovary (CHO) cell lines, W138, BHK, COS-7, 293, HepG2,NIH3T3, RIN and MDCK cell lines. In addition, a host cell type isoptionally chosen that modulates the expression of the insertedsequences, or modifies and processes the gene product in the specificfashion desired. Such modifications (e.g., glycosylation) and processing(e.g., cleavage) of protein products may be important for the functionof the encoded protein.

Different host cells have characteristic and specific mechanisms for thepost-translational processing and modification of proteins. Appropriatecell lines or host systems are preferably chosen to ensure the correctmodification and processing of the foreign protein expressed. Expressionvectors for use in mammalian cells ordinarily include an origin ofreplication (as necessary), a promoter located in front of the gene tobe expressed, along with any necessary ribosome binding sites, RNAsplice sites, polyadenylation site, and transcriptional terminatorsequences. The origin of replication is optionally provided either byconstruction of the vector to include an exogenous origin, such as maybe derived from SV40 or other viral (e.g., Polyoma, Adeno, VSV, BPV)source, or may be provided by the host cell chromosomal replicationmechanism. If the vector is integrated into the host cell chromosome,the latter is often sufficient.

The promoters are optionally derived from the genome of mammalian cells(e.g., metallothionein promoter) or from mammalian viruses (e.g., theadenovirus late promoter; the vaccinia virus 7.5K promoter). Further, itis also possible, and may be desirable, to utilize promoter or controlsequences normally associated with the desired gene sequence, providedsuch control sequences are compatible with the host cell systems.

A number of viral based expression systems are optionally utilized, forexample, commonly used promoters are derived from polyoma, Adenovirus 2,Adenovirus 5, cytomegalovirus and Simian Virus 40 (SV40). The early andlate promoters of SV40 virus are useful because both are obtained easilyfrom the virus as a fragment which also contains the SV40 viral originof replication. Smaller or larger SV40 fragments are also optionallyused, preferably provided there is included the approximately 250 bpsequence extending from the HindIII site toward the BglI site located inthe viral origin of replication.

In cases where an adenovirus is used as an expression vector, the codingsequences are optionally ligated to an adenovirustranscription/translation control complex, e.g., the late promoter andtripartite leader sequence. This chimeric gene may then be inserted inthe adenovirus genome by in vitro or in vivo recombination. Insertion ina non-essential region of the viral genome (e.g., region E1 or E3) willresult in a recombinant virus that is viable and capable of expressingproteins in infected hosts.

Specific initiation signals are optionally provided for efficienttranslation of the claimed isolated nucleic acid coding sequences. Thesesignals illustratively include the ATG initiation codon and adjacentsequences. Exogenous translational control signals, including the ATGinitiation codon, may additionally need to be provided. One of ordinaryskill in the art would readily be capable of determining this need andproviding the necessary signals. It is well known that the initiationcodon must be in-frame (or in-phase) with the reading frame of thedesired coding sequence to ensure translation of the entire insert.These exogenous translational control signals and initiation codons areoptionally of a variety of origins, both natural and synthetic. Theefficiency of expression is optionally enhanced by the inclusion ofappropriate transcription enhancer elements or transcriptionterminators.

In eukaryotic expression, one will also typically desire to incorporateinto the transcriptional unit an appropriate polyadenylation site if onewas not contained within the original cloned segment. Typically, thepoly A addition site is placed about 30 to 2000 nucleotides downstreamof the termination site of the protein at a position prior totranscription termination.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. Illustratively, cell lines that stably expressconstructs encoding proteins are engineered. Rather than usingexpression vectors that contain viral origins of replication, host cellsare optionally transformed with vectors controlled by appropriateexpression control elements (e.g., promoter, enhancer, sequences,transcription terminators, polyadenylation sites, etc.), and aselectable marker. Following the introduction of foreign DNA, engineeredcells are illustratively allowed to grow for 1-2 days in an enrichedmedium, and then are switched to a selective medium. The selectablemarker in the recombinant plasmid confers resistance to the selectionand allows cells to stably integrate the plasmid into their chromosomesand grow to form foci, which in turn can be cloned and expanded intocell lines.

A number of selection systems are optionally used, including, but notlimited, to the herpes simplex virus thymidine kinase,hypoxanthine-guanine phosphoribosyltransferase and adeninephosphoribosyltransferase genes, in tk⁻, hgprt⁻ or aprt⁻ cells,respectively. Also, antimetabolite resistance is optionally used as thebasis of selection for dhfr, which confers resistance to methotrexate;gpt, which confers resistance to mycophenolic acid; neo, which confersresistance to the aminoglycoside G-418; and hygro, which confersresistance to hygromycin. It is appreciated that numerous otherselection systems are known in the art that are similarly operable inthe present invention.

It is contemplated that the isolated nucleic acids of the disclosure areoptionally overexpressed; i.e. expressed in increased levels relative toits natural expression in cells of its indigenous organism, or evenrelative to the expression of other proteins in the recombinant hostcell. Such overexpression is illustratively assessed by a variety ofmethods, including radio-labeling and/or protein purification. However,simple and direct methods are preferred, for example, those involvingSDS/PAGE and protein staining or immunoblotting followed by quantitativeanalyses such as densitometric scanning of the resultant gel or blot. Aspecific increase in the level of the recombinant protein or peptide incomparison to the level in natural human cells is indicative ofoverexpression, as is a relative abundance of the specific protein inrelation to the other proteins produced by the host cell.

Further aspects of the present disclosure concern the purification, andin particular embodiments, the substantial purification, of an encodedprotein or peptide. The term “purified” or “isolated” protein or peptideas used herein, is intended to refer to a composition, isolatable fromother components, wherein the protein or peptide is purified to anydegree relative to its naturally-obtainable state, i.e., in this case,relative to its purity within a cell of a tick salivary gland. Apurified protein or peptide, therefore, also refers to a protein orpeptide free from the environment in which it may naturally occur.

Generally, “purified” or “isolated” will refer to a protein or peptidecomposition that has been subjected to fractionation to remove variousother components, and which composition substantially retains itsexpressed biological activity. Where the term “substantially” purifiedis used, this designation will refer to a composition in which theprotein or peptide forms the major component of the composition, such asconstituting about 50% or more of the proteins in the composition.

Various methods for quantifying the degree of purification of theprotein or peptide will be known to those of skill in the artparticularly in light of the present disclosure. These include, forexample, determining the specific activity of an active fraction, orassessing the number of polypeptides within a fraction by SDS/PAGEanalysis. A preferred method for assessing the purity of a fraction isto calculate the specific activity of the fraction, to compare it to thespecific activity of the initial extract, and to thus calculate thedegree of purity, herein assessed by a “-fold purification number”. Theactual units used to represent the amount of activity will, of course,be dependent upon the particular assay technique chosen to follow thepurification and whether or not the expressed protein or peptideexhibits a detectable activity.

Various techniques suitable for use in protein purification will be wellknown to those of skill in the art. These include, for example,precipitation with ammonium sulphate, polyethylene glycol, antibodiesand the like or by heat denaturation followed by centrifugation,chromatography steps such as ion exchange and the like, gel filtration,reverse phase, hydroxylapatite and affinity chromatography isoelectricfocusing gel electrophoresis and combinations of such and othertechniques. As is generally known in the art, it is believed that theorder of conducting the various purification steps may be changed, orthat certain steps may be omitted, and still result in a suitable methodfor the preparation of a substantially purified protein or peptide.

There is no general requirement that the protein or peptide always beprovided in their most purified state. Indeed, it is contemplated thatless substantially purified products will have utility in certainembodiments. Partial purification is illustratively accomplished byusing fewer purification steps in combination, or by utilizing differentforms of the same general purification scheme. For example, it isappreciated that a cation-exchange column chromatography performedutilizing an HPLC apparatus will generally result in a greater-foldpurification than the same technique utilizing a low pressurechromatography system. Methods exhibiting a lower degree of relativepurification may have advantages in total recovery of protein product orin maintaining the activity of an expressed protein.

It is known that the migration of a polypeptide can vary, sometimessignificantly, with different conditions of SDS/PAGE (Capaldi et al.,Biochem. Biophys. Res. Comm., 76:425, 1977). It will, therefore, beappreciated that under differing electrophoresis conditions the apparentmolecular weights of purified or partially purified expression productsmay vary.

It is recognized in the art that several phospholipase A₂ proteins gaintheir full activity when bound to phospholipid micelles or membranes, aneffect known as interfacial activation (Scott, D. L., and Sigler, P. B.(1994) Adv. Protein Chem. 45, 53-88; Arni, R. K., and Ward, R. J. (1996)Toxicon, 34, 827-841; Berg, O. G., et al. (2001)Chem. Rev., 101,2613-2654) the contents of each of which are incorporated herein byreference. The N-terminal portion of phospholipase A₂ is believed to beessential for mediating this interaction (Qin, S., Pande, et al., (2004)J. Mol. Biol., 344, 71-89). Additionally, the catalytic histidine(His⁴⁸) and functionally important tyrosine (Tyr⁶⁹) on the humanphospholipase A₂ are encompassed by structural regions that areimportant for enzymatic substrate binding and catalytic activity (humanphospholipase A₂ numbering). As such, the invention envisions fragmentsof PLA2 that allow for individual or combined activities such asmembrane binding or catalytic function that are individually isolated orcombined free of non-essential regions of the inventive PLA2 protein ornucleic acid sequence. The invention also envisions isolation offragments of PLA2 encompassing regions with activities unique tophospholipase A₂ from A. americanum.

An inventive process illustratively includes isolation of PLA2 proteinor nucleic acid sequence from a host cell or host cell medium. Methodsof protein isolation illustratively include column chromatography,affinity chromatography, gel electrophoresis, filtration, or othermethods known in the art. In a preferred embodiment PLA2 protein isexpressed with a tag operable for affinity purification. A preferred tagis a 6×His tag. A 6×His tagged inventive protein is illustrativelypurified by Ni-NTA column chromatography or using an anti-6×His tagantibody fused to a solid support. (Geneway Biogech, San Diego, Calif.)Other tags and purification systems are similarly operable.

It is appreciated that an inventive protein is optionally not tagged. Inthis embodiment and other embodiments purification is optionallyachieved by methods known in the art illustratively includingion-exchange chromatography, affinity chromatography using anti-PLA2antibodies, precipitation with salt such as ammonium sulfate,streptomycin sulfate, or protamine sulfate, reverse phasechromatography, size exclusion chromatography such as gel exclusionchromatography, HPLC, immobilized metal chelate chromatography, or othermethods known in the art. One of skill in the art may select the mostappropriate isolation and purification techniques without departing fromthe scope of this invention.

An inventive PLA2 protein or fragment thereof is optionally chemicallysynthesized. Methods of chemical synthesis have produced proteinsgreater than 600 amino acids in length with or without the inclusion ofmodifications such as glycosylation and phosphorylation. Methods ofchemical protein and peptide synthesis illustratively include solidphase protein chemical synthesis. Illustrative methods of chemicalprotein synthesis are reviewed by Miranda, L P, Peptide Science, 2000,55:217-26 and Kochendoerfer G G, Curr Opin Drug Discov Devel. 2001;4(2):205-14, the contents of each of which are incorporated herein byreference.

PLA2 proteins of this invention are optionally characterized byimmunological measurements including, without limitation, western blot,macromolecular mass determinations by biophysical determinations,SDS-PAGE/staining, HPLC and the like, antibody recognition assays, cellviability assays, apoptosis assays, and assays to infer immuneprotection or immune pathology by adoptive transfer of cells, proteinsor antibodies.

An inventive PLA2 protein of the present invention is optionallymodified to increase its immunogenicity. In a non-limiting example, theantigen is coupled to chemical compounds or immunogenic carriers,provided that the coupling does not interfere with the desiredbiological activity of either the antigen or the carrier. For a reviewof some general considerations in coupling strategies, see Antibodies, ALaboratory Manual, Cold Spring Harbor Laboratory, ed. E. Harlow and D.Lane (1988) the contents of which are incorporated herein by referencefor this and other relevant teaching. Useful immunogenic carriers knownin the art, include, without limitation, keyhole limpet hemocyanin(KLH); bovine serum albumin (BSA), ovalbumin, PPD (purified proteinderivative of tuberculin); red blood cells; tetanus toxoid; choleratoxoid; agarose beads; activated carbon; or bentonite. Useful chemicalcompounds for coupling include, without limitation, dinitrophenol groupsand arsonilic acid.

The inventive PLA2 protein is optionally modified by other techniques,illustratively including denaturation with heat and/or SDS.

A PLA2 protein of the present invention is optionally used in the formof a pharmaceutically acceptable salt. Suitable acids and bases whichare capable of forming salts with the polypeptides of the presentinvention are well known to those of skill in the art and includeinorganic and organic acids and bases.

In another aspect, the invention provides a therapeutic composition andmethods for treating humans and/or animals with Lyme disease orinfection with a spirochete. The therapeutic composition contains aninventive PLA2 protein, nucleic acid sequence, or fragment thereof asdescribed above and a suitable pharmaceutical carrier.

The proteins and nucleic acid sequences or anti-sense sequences of theinvention, alone or in combination with other antigens, antibodies,nucleic acid sequences or anti-sense sequences are optionally used intherapeutic compositions and in methods for treating humans and/oranimals with spirochete or other bacterial disease illustrativelyincluding Lyme Disease. For example, one such therapeutic composition isformulated to contain a carrier or diluent and one or more PLA2 proteinsor protein fragments of the invention. Suitable pharmaceuticallyacceptable carriers facilitate administration of the proteins but arephysiologically inert and/or nonharmful.

Carriers are optionally selected by one of skill in the art. Exemplarycarriers include sterile water or saline, lactose, sucrose, calciumphosphate, gelatin, dextran, agar, pectin, peanut oil, olive oil, sesameoil, and water. Additionally, the carrier or diluent may include a timedelay material, such as glycerol monostearate or glycerol distearatealone or with a wax. In addition, slow release polymer formulations canbe used.

Optionally, the inventive composition contains conventionalpharmaceutical ingredients such as preservatives or chemicalstabilizers. Suitable ingredients operable herein include, for example,casamino acids, sucrose, gelatin, phenol red, N-Z amine, monopotassiumdiphosphate, lactose, lactalbumin hydrolysate, and dried milk.

Alternatively, or in addition to the PLA2 proteins of the presentinvention, other agents useful in treating Lyme disease, e.g.,antibiotics or immunostimulatory agents and cytokine regulationelements, are expected to be useful in reducing or eliminating diseasesymptoms. Agents operable herein to suppress or counteract the immunesuppressants released by the tick vector or the spirochete preferablyact to assist the natural immunity of the infected human or animal.Thus, such agents optionally operate in concert with the therapeuticcompositions of this invention. The development of therapeuticcompositions containing these agents is within the skill of one in theart in view of the teachings of this invention.

According to the invention, a human or an animal is optionally treatedfor Lyme Disease or other spirochete or bacterial infection byadministering an effective amount of such a therapeutic composition. An“effective amount” is preferably between about 0.05 to about 1000 μg/mLof an PLA2 protein. A suitable dosage is preferably about 1.0 mL of suchan effective amount. Such a composition is optionally administered 1-3times per day over a 1 day to 12 week period. However, suitable dosageadjustments may be made by the attending physician or veterinariandepending upon the age, sex, weight, composition pharmacokinetics,composition pharmakodynamics, and general health of the subject.Preferably, such a composition is administered parenterally, preferablyintramuscularly or subcutaneously. However, an inventive composition isoptionally formulated to be administered by any other suitable route,including orally or topically.

It is appreciated that the efficacy of the present invention is readilydetermined with respect to altering bacterial organism function bygrowing the organisms in culture and placing a spatially controlled andknown amount of the inventive PLA2 into contact with the organismculture and then measuring an inhibition zone around the phospholipase.With culture efficacy, non-human animals infected with the bacterialorganism are then administered the inventive phospholipase by knownmethods such as intravenous or intramuscular injection. Bacterialorganism titers relative to a control provide in vivo efficacy anddosing regimes extendible to humans.

Methods involving conventional biological techniques are describedherein. Such techniques are generally known in the art and are describedin detail in methodology treatises such as Molecular Cloning: ALaboratory Manual, 2nd ed., vol. 1-3, ed. Sambrook et al., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; and CurrentProtocols in Molecular Biology, ed. Ausubel et al., Greene Publishingand Wiley-Interscience, New York, 1992 (with periodic updates).Immunological methods (e.g., preparation of antigen-specific antibodies,immunoprecipitation, and immunoblotting) are described, e.g., in CurrentProtocols in Immunology, ed. Coligan et al., John Wiley & Sons, NewYork, 1991; and Methods of Immunological Analysis, ed. Masseyeff et al.,John Wiley & Sons, New York, 1992. The contents of each of the hereinincluded references are incorporated herein by reference in theirentirety.

The invention is further described by reference to the followingdetailed examples, wherein the methodologies are as described below.These examples are not meant to limit the scope of the invention thathas been set forth in the foregoing description. Variations within theconcepts of the invention are apparent to those skilled in the art.

EXAMPLE 1

The cDNA of full-length A. americanum PLA2 is cloned from an A.americanum cDNA library and subcloned into the pET-21a(+) vector(Novagen, Madison, Wis.). The cDNA library is created from genomic DNAfrom A. americanum using the CloneMiner™ cDNA Library Construction Kitavailable from Invitrogen (Carlsbad, Calif.). Amplification and cloningof plasmids containing the cDNA for PLA2 is performed essentially asdescribed by Wijewickrama, G T, et al., J. Biol. Chem., 2006;281(43):32741, the contents of which are incorporated herein byreference. All constructs are transformed into DH5 cells for plasmidisolation, and their DNA sequences are verified. E. coli strain BL21(DE3) was used as a host for the protein expression.

EXAMPLE 2

Isolation and purification of PLA2. PLA2 is purified from E. coli BL21(DE3). The plasmid encoding PLA2 adds an amino-terminal His tag forsubsequent purification. Optionally, the His tag is cleavable byincorporation of a cleavage sequence for enzymes such as trypsin, factorXa, or thrombin. The bacteria are grown overnight at 37° C. in 2 litersof LB broth supplemented with 100 mg/liter of ampicillin; harvested bycentrifugation; suspended in 30 ml of 10 mM Tris.HCl (THCl), pH 8.3; andsonicated for 15 min on ice. The cell debris is removed bycentrifugation at 20,000×g for 15 min, and the supernatant is loadedonto a Ni-NTA agarose (Qiagen) column (1.5×3 cm). The column is washedwith 50 ml of 1.0 M NaCl in THCl, and the protein eluted with a 40-mllinear gradient of 0-0.25 M imidazole in THCl. Recombinant protein isidentified by SDS/PAGE, and peak fractions are pooled. The protein isdialyzed against 3 liters of THCl at 4° C. overnight and loaded on aDEAE Sepharose column (1.5×5 cm) equilibrated with THCl. Protein iseluted with a 60-ml linear gradient of 0-0.15 M NaCl. The purifiedprotein is again dialyzed against 3.5 liters of THCl. Purifiedrecombinant PLA2 is free of contaminating proteins as assessed byCoomassie blue-stained SDS/PAGE. All reagents and glassware used fortoxin purification and biological assays are pyrogen-free.

EXAMPLE 3

Evaluation of PLA2 protein regulation of B. burgdorferi viability.Analyses of PLA2 protein is performed essentially as described by Ledin,K. E., et al., Med. Vet. Entomol., 2005; 19(1):90-95, the contents ofwhich are incorporated herein by reference. Frozen stocks of low-passageB31 B. burgdorferi isolates (Shelter Island, N.Y.) are reconstituted inBSK-H culture medium and maintained at 35° C. to log phase (Piesman,1993). Spirochete cultures are diluted with BSK-H to a density of4.7×10⁷ viable spirochetes per mL as counted in a Petroff-Hauser chamberunder dark-field microscopy, and distributed in 15 μL aliquots into 0.7mL tubes for a total of 7.05×10⁵ spirochetes per tube. A mean startingconcentration of 155 live spirochetes per field is assessed prior to theaddition of specific treatments. Each 15 μL aliquot of spirochetes inBSK-His treated with either 15 μL of sterile PBS, 15 μL of 1.33 mg/mLpilocarpine (based on the mid-range of pilocarpine found in previousanalysis of tick saliva, (Ribeiro et al., 2004) in sterile PBS, or 15 μLof tick saliva, and incubated at 35° C. 10 μL samples taken from each of10 prepared cultures and assessed for spirochete survival at 0, 24 and48 h. Time points for spirochete survival assessment are chosen based onprevious observations of post-feeding decreases in spirochete-positiveticks (Ryder et al., 1992).

Spirochete survival is evaluated by enumeration of spirochetes stainedwith the live/dead BacLight Viability Kit (Molecular Probes, Eugene,Oreg.). Briefly, fluorescence stains SYTO 9 (live stain) and propidiumiodide (dead stain) are combined at a 1:100 dilution in sterile PBS(Invitrogen, Grand Island, N.Y.). A total of 0.5 μL of this solution ismixed with 10 μL from each spirochete culture. Stained cultures aresuspended on a standard glass slide under a 22×22 mm cover slip andviewed at 100× magnification through FITC and rhodamine filters. Liveand dead spirochetes are counted from 10 randomly chosen high-powerfields per slide. Results are compared with the saliva of I. scapularisas a negative control. PLA2 protein in buffered saline is used assynthetic tick saliva.

Using this method live spirochetes stain green (FIGS. 1A and B) and canbe readily visualized and distinguished from injured and deadspirochetes that stain red (FIGS. 1C and D). This analysis (FIG. 2)indicates that treatment with PLA2 protein significantly reduces theaverage number of live spirochetes at both 24 h (mean=39.6) and 48 h(mean=20.1) compared with both pilocarpine at 24 and 48 h (82.1/58.2)and PBS alone at 24 and 48 h (133/112) (all P<0.001). Furthermore,average counts for PLA2 are lower than average counts for I. scapularistreatment at 24 and 48 h (100.2/75.0, P<0.001 for both time points). Incontrast, I. scapularis does not yield significantly differentspirochete counts than pilocarpine at both time points, whiledemonstrating a significant killing effect compared to PBS at 24 and 48h (P<0.001 for both).

EXAMPLE 4

Incubation with trypsin eliminates A. americanum borreliacidal activity.Colony-produced, pathogen-free A. americanum adults are obtained fromthe Oklahoma State University Tick Rearing Facility (Stillwater, Okla.).Colony maintained, pathogen-free I. scapularis adults are obtained fromthe Tick Biology and Ecology Laboratory of the CDC (Fort Collins,Colo.). To collect saliva, adult New Zealand rabbits (Western OregonRabbit Company) are infested with 20-30 pairs of adult A. americanum orI. scapularis ticks, and saliva is collected beginning at the rapidengorgement phase for each tick species as previously described (Ledinet al., 2005). Saliva is then aliquoted and frozen at −80° C. until use.

Whole saliva is incubated with B. burgdorferi in the presence of 10 μgof bovine trypsin essentially as described in Zeidner, N, et al.,incorporated herein by reference. A borreliacidal factor in Amblyommaamericanum saliva is associated with phospholipase A₂ activity, Zeidner,N, et al., Experimental Parasitology, 2009; 121(4):370-5, the entirecontents of which are incorporated herein by reference as if eachcharacter were explicitly laid out herein. The results presented in FIG.3 demonstrate that the borreliacidal factor found in A. americanumsaliva is of protein origin.

EXAMPLE 5

Comparative PAGE analysis of A. americanum and I. scapularis salivademonstrate unique protein bands. Saliva of A. americanum and I.scapularis are analyzed by SDS-PAGE by methods known in the art anddescribed by Zeidner, et al. 2009. Protein bands of approximately 120,70, 31, 26, 23, and 21 kDa are identified in A. americanum saliva thatare not recognized in saliva from I. scapularis (FIG. 4). A proteinrunning at approximately 58-61 kDa is very prominent in A. americanumsaliva when compared to I. scapularis (asterisks/arrow, FIG. 4).

EXAMPLE 6

Gel filtration fractionation of A. americanum saliva produces individualfractions with borreliacidal activity. Saliva is fractionated into 11molecular weight pools by gel filtration and then tested by aborreliacidal assay as described by Zeidner, et al. 2009. Briefly, A.americanum saliva is size fractionated on a Superdex 200 column run with25 mM NaCl dissolved in 5 mM HEPES (pH 7.6) at a flow rate of 0.5 ml perminute. The separation is monitored at 280 nM. Fractions are collectedat 1 minute intervals into siliconized eppendorf tubes. Each collectedfraction is subsequently concentrated to 100 μl using a SpeedVac. 50 μlaliquots of the fractions are pooled according to the major molecularweight peaks at between 2 and 10 fractions per pool. Each pool issubsequently concentrated to 50 μl using Micron-3K filters (Millipore,Billerica, Mass.). Pools and then the individual component fractions ofpools that demonstrate activity are then bioassayed for borreliacidalactivity in vitro substantially as described in Example 4.

As noted in FIG. 5A, pools number 3 and 4 demonstrate significantborreliacidal activity compared to BSK-H controls. Only 69.4+/−7.3percent of control spirochetes survive after 24 hrs of exposure to pool3 (p<0.025), while exposure to pool 4 virtually eliminates all livespirochetes at 24 hrs post-incubation (2.7+/−0.8 percent of controls,p<0.001). Borreliacidal activity is not noted with any other pools whencompared to media controls (FIG. 5A). Pool 3 demonstrates a highermolecular weight shoulder associated with pool 4 (data not shown),suggesting that the activity could result from a protein that overlapsboth pools.

Fractions of pool 4 are further examined for borreliacidal assay (FIG.5B). Two fractions demonstrate significant killing activity whencompared to media controls at 24 hrs. Although quite diminished inactivity when compared to the original pool 4, fractions numbered 2(spirochete survival, 63.3+/−5 percent of control, p<0.001) and 5(spirochete survival, 72.6+/−8 percent of control, p<0.05) demonstratesignificant killing activity (FIG. 5B). Fractions numbered 1, 3 and 4 donot demonstrate any significant killing of spirochetes compared to mediacontrols (FIG. 5B).

EXAMPLE 7

Comparative 2D gel analysis of active and inactive saliva fractionsderived from gel filtration identifies prominent peptides includingphospholipase A₂. Individual fractions of pool 4 from Example 6(numbered 1-5) are analyzed by 2-dimensional gel electrophoresis. Asnoted in FIG. 6A, a series of prominent peptides or protein arelocalized in the 38 kDa range (FIG. 6A, arrow) and in the 58-62 kDamolecular weight range (circle) when borreliacidal fractions numbered 2and 5 are combined and analyzed by two-dimensional PAGE. When comparedto a 2-dimensional PAGE which includes fractions 1, 3 and 4 (FIG. 6B),no protein or peptides are localized to these regions, suggesting theborreliacidal activity of A. americanum saliva represents proteins orpeptides within this 38-62 kDa molecular weight range (FIG. 6A). Theisoelectric point (pI) of these proteins is approximately 4.5-5.0.

EXAMPLE 8

Inhibition of A. americanum borreliacidal activity using oleyloxyethylphosphorylcholine (OPC) identifies phospholipase A₂ as an activecomponent of A. americanum saliva. The borreliacidal assay of Example 6is performed comparing saliva alone with saliva plus the phospholipaseA₂ inhibitor OPC. At 24 hrs post incubation with saliva alone, theborreliacidal effect of A. americanum saliva (percent control livespirochetes, 30+/−7) is completely eliminated with concentrations of OPCranging from 5-40 μM (percent control live spirochetes, 76-78, p<0.0001,FIG. 7). The percent control live spirochete numbers after incubationwith saliva plus OPC are not significantly different than culturecontrols (79, p=0.45). As noted in FIG. 7, no titration effect isobserved using concentrations of OPC ranging from 5-40 μM. These datademonstrate that the borreliacidal activity of A. americanum saliva isdependent on the enzymatic activity of phospholipase A₂.

The invention is hereby described with relation to the followingreferences and those otherwise identified in the instant specification.Each reference is incorporated herein by reference as if each were laidout explicitly in its entirety in the instant specification includingboth text and figures. Each reference is incorporated for the individualpoint referred to in the specification as well as for all informationcontained therein and not explicitly identified in the specification.All references are representative of the knowledge of a person of skillin the art and illustrate other aspects of the present invention asenvisioned by the inventors.

It is appreciated that all reagents are obtainable by sources known inthe art unless otherwise specified. Methods of nucleotide amplification,cell transfection, and protein expression and purification are similarlywithin the level of skill in the art.

Patent applications and publications mentioned in the specification areindicative of the levels of those skilled in the art to which theinvention pertains. These applications and publications are incorporatedherein by reference to the same extent as if each individual applicationor publication was specifically and individually incorporated herein byreference.

The foregoing description is illustrative of particular embodiments ofthe invention, but is not meant to be a limitation upon the practicethereof. The following claims, including all equivalents thereof, areintended to define the scope of the invention.

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The invention claimed is:
 1. A process of modulating Borreliaburgdorferi infection of a subject comprising administering to a subjecta therapeutic composition comprising a suitable pharmaceutical carrierand an isolated phospholipase A2 protein derived from Amblyommaamericanum.
 2. The process of claim 1 wherein said therapeuticcomposition is administered at from about 0.05 to about 10,000micrograms/milliliter.
 3. The process of claim 1 wherein saidtherapeutic composition is administered between one to three times perday.
 4. The process of claim 1 wherein said administering treats LymeDisease in said subject.